The overall, long-term objective of this project is elucidation of the biochemical mechanisms underlying granulocyte proliferation and differentiation. Our approach is to define, at the molecular level, the regulatory role of colony stimulating activity (CSA), the presumptive primary inducer of normal granulopoietic and leukemic cell differentiation. Implementation of this approach centers around development of convenient and quantitative radioimmunoassays (RIA) and immunocytofluorescence (IF) procedures for studying the CSA-dependent synthesis of granulocyte-specific proteins in a readily accessible animal model, the mouse. In particular, we are studying the protein myeloperoxidase (MPO) and lactoferrin (LF). Each is synthesized at a different period of the granulocyte maturation sequence, and consequently, each is initially associated with a different recognizable cell type (promyelocyte and myelocyte, respectively). We have employed a liquid culture method using slide chambers (Blood 50:289-302, 1977) to study the effect of increasing concentrations of CSA on the average proportion of neutrophilic granulocyte clone cells containing LF. The data indicate that CSA in higher concentrations delays the initiation of LF synthesis by these cells. Post-vinblastine (VB) regeneration of the progressively more mature granulopoietic cells was temporally correlated with marrow production of humoral activity that stimulated in vitro granulocyte proliferation. The stimulating activity produced by freshly explanted mouse marrow during 8 hrs of media conditioning was assayed in 3 day slide chamber cultures. Marrow production of humoral factors that stimulated in vitro granulopoiesis increased 8 fold above normal one day post-VB, coincident with the nadir of CFUGM, promyelocytes and monocytes. Marrow stimulator production began to decline as CFUGM and monocytes recovered on days 2 and 3 post VB. By the fourth day post-VB, marrow production of stimulator was below normal. While promyelocytes had recovered to near normal numbers at this time, mature LF containing neutrophils remained markedly decreased. These observations do not support the concept that mature neutrophils play an important role in suppressing the production of humoral granulopoietic stimulators.